ABSTRACT Mitochondrial diseases manifesting as encephalopathies occur at a rate of 1 in 5000 live births and are often fatal by ~5 years old. Mitochondrial diseases are respiratory chain disorders in which the mitochondria are no longer operating efficiently to produce ATP, usually due to a problem with one or more components of the electron transport chain (ETC). Fortunately, genetic sequencing has identified a large number of the mutations in mitochondrial or nuclear DNA which cause these encephalomyopathies. However, in most cases there is still no clear metabolic link between the genetic defect and the neuropathology, and very few effective treatments. The innovative studies described in this proposal are expected to reveal a novel metabolic link between reduced ETC activity and neural pathology. Previously, we have detected a new post-translational modification of proteins, S-(2-succino)cysteine (2SC), which is formed by reaction of the Krebs cycle intermediate fumarate with reactive cysteine residues in protein. Both fumarate and succination of proteins are increased in adipocytes in diabetes, disturbing protein function and turnover. The increase in fumarate develops as a result of excess fuel supply, accumulation of NADH, and feedback inhibition of the Krebs cycle. In a novel, lateral extension of these observations we propose that a similar inhibition of the ETC, e.g. in Complex I deficiency during Leigh Syndrome, would result in increased NADH, fumarate and succination in mitochondrial disease. In Preliminary Studies, we demonstrate that increased succination of proteins is detectable on several proteins in the brainstem of a mouse model of Leigh syndrome (Ndufs4 knockout (KO) mouse) in association with neurodegeneration. We hypothesize that mitochondrial stress results in the accumulation of fumarate and that succination alters protein structure or function contributing to disease pathology. We will confirm this in Specific Aim 1. We have identified several succinated targets already and we plan to mechanistically address how succination of these leads to further reductions in mitochondrial function in Specific Aim 2. In Specific Aim 3 we will use a molecular strategy to distinguish the bioenergetic defect from protein succination and investigate therapeutic strategies designed to reduce fumarate and succination leading to improvements in mitochondrial function and the disease phenotype. Overall, these foundational studies will demonstrate that succination is a mechanistic link between mitochondrial stress and neuropathology, with important implications for the elucidation of novel therapeutic avenues for the treatment of mitochondrial diseases.